ABSTRACT Full-length CD33 (CD33FL) is characterized by a membrane-distal V-set immunoglobulin (Ig)-like domain and a membrane-proximal C2-set Ig-like domain in its extracellular portion. Consistent with a myeloid differentiation antigen, CD33FL is widely expressed on malignant cells in patients with myeloid neoplasms, most notably acute myeloid leukemia (AML), where it is found on at least a subset of the AML blast cells in almost all cases and possibly leukemic stem cells in some. Several randomized trials with the CD33 antibody-drug conjugate, gemtuzumab ozogamicin (GO), have demonstrated improved survival in subsets of patients with AML, validating CD33FL as the first and, so far, only therapeutic target for immunotherapy of AML. In addition to CD33FL, we have recently demonstrated mRNA expression of a CD33 splice variant that lacks exon 2 (CD33?E2) in all tested AML patient specimens. CD33?E2 contains the C2-set but not V-set domain. Since all available CD33 antibody-based therapeutics recognize the immune-dominant V-set domain, CD33?E2 is not targetable with any current agent. However, we have very recently raised murine antibodies against the human C2-set domain of CD33 and demonstrate that antibodies recognizing CD33?E2 alone or CD33?E2 and CD33FL (CD33?E2+FL) can indeed be generated. Because of greater target antigen density for antibodies recognizing CD33?E2+FL as well as more membrane-proximal binding compared to antibodies recognizing the V-set domain, we hypothesize that CD33?E2+FL antibodies could provide a novel means of CD33 immunotherapy that is superior to current therapeutics. To begin testing this idea, we propose preclinical studies to determine the value of CD33?E2+FL antibodies for the treatment AML in carefully-controlled in vitro and in vivo analyses. Our research will take advantage of transgenic, humanized mice to directly generate fully human antibodies that are potential therapeutics. A second innovative aspect of our research is the use of mice with humanized immune system to enable direct in vivo assessments of our antibodies. Our studies are expected to provide insight into the therapeutic potential of C2-set domain-specific antibodies recognizing CD33?E2+FL. Successful completion of the proposed work will result in clinical development candidates that would require little, if any, optimization to advance to human trials. Overall, our work is anticipated to have an important positive impact because it may lead to a new treatment option for patients with AML and other myeloid neoplasms for whom outcomes continue to be unsatisfactory.